We have determined the three-dimensional structure of d(GCATATGATAG) .d(CTATCATATGC), a DNA duplex found in the promoter region of a gene regulating sporulation in a Bacillus Subtilis mother cell, utilizing to-dimensional nuclear Overhauser effect (2D NOE) spectra. We assigned nearly all of the duplex proton resonances using 2D NOE spectra, obtained at four different mixing times, and double quantum-filtered COSY (2QF-COSY) spectra. We calculated interproton distances from 2D NOE intensities in a very conservative fashion by running the complete relaxation matrix program MARDIGRAS hundreds of times using 2D NOE spectra for exchangeable protons and for nonexchangeable protons recorded at different mixing times, assuming different overall correlation times and several different starting structures. The 435 distance restraints we calculated, were used with two different structural refinement methods: restrained molecular dynamics (rMD) in cartesian coordinate space and restrained Monte Carlo calculations (rMC) in internal coordinates. Our refinements using the different procedures and different starting structures resulted in convergence to essentially the same structure (<0.8 A for nonterminal residues), lending us confidence that our final structure is defined by experimental restraints and does not depend significantly on the refinement method or variables used. Comparison of the experimental 2D NOE intensities and intensities calculated for the refined structures via the conventional R factors and sixth-root R factors, indicated that our converged structures also fit the experimental NOE data very well. Thus, our converged structure provides an accurate representation of the time- averaged structure of our duplex in solution. Some helical parameters of our refined time-averaged structure, most notably the large negative X-dispacement, exhibit some characteristics of A-DNA rather than being strictly in the B-DNA family of structures as one might expect for a DNA duplex in solution. As with the TG/CA steps in other duplex DNA sequences studied in our laboratory, the two TG/CA steps in our undecamer have a positive roll with T6-G7 exhibiting the large roll, and consequently the helix axis is bent. We might speculate that bending in the free solution may confer some advantage in binding of the promoter to the Sigma-K transcription factor. Visual inspection of the DNA models with the program MidasPlus before, during, and after simulations is an essential part of the process of refining and analyzing the final NMR structure. We also used MidasPlus to visualize the trajectories of the molecular dynamics simulations. This is a critical step in the analysis of rMD runs. We used the Computer Graphics Laboratory resources to generate the DNA models that were helpful in assigning the 2D NOE intensities and that later we used as starting points for dynamic simulations.